CN109232706A - 一类三萜-寡糖偶联物及其应用 - Google Patents
一类三萜-寡糖偶联物及其应用 Download PDFInfo
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- CN109232706A CN109232706A CN201811036386.6A CN201811036386A CN109232706A CN 109232706 A CN109232706 A CN 109232706A CN 201811036386 A CN201811036386 A CN 201811036386A CN 109232706 A CN109232706 A CN 109232706A
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- A—HUMAN NECESSITIES
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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Abstract
本发明公开了结构式如下式所示的三萜‑寡糖偶联物:
Description
技术领域
本发明涉及了一类三萜-寡糖偶联物及其应用,即其在预防或治疗流感中的用途。
背景技术
流感是由流感病毒(Influenza virus)引起的一种急性、传染性呼吸系统疾病。根据其内部核蛋白(NP)和基质蛋白(M)的抗原性不同,流感病毒可分为A型,B型,C型和D型。A型(又称甲型)流感病毒大规模流行可引起极高的发病率和死亡率,严重威胁着人类的健康(Virology Journal.2007,4,1-5)。A型流感病毒在二十世纪主要引起了三次大型流感,即1918年的H1N1,1957年的H2N2以及1968年的H3N2,共造成约5000万人死亡(EmergingInfectious Diseases.2006,12,9-14;Journal of the American Medical Association,2007,18,2025-2027)。2009年甲型流感也是由H1N1流感病毒引起(New England Journalof Medicine.2009,370,1335-1342),其传播之迅速,引起了世界的关注。据统计,全世界平均每年有30-50万人死于流感(Southern Medical Journal.2007,57,1-60)。
迄今,FDA批准的抗流感药物主要有两类;第一类,达菲(Oseltamivir)和乐感清(Zanamivir)主要抑制流感病毒的神经氨酸酶(NA),阻断流感病毒从感染细胞中释放出来(Nature Medicine.2004,10,82-87;Journal of the Americ an Chemical Society1997,119,681-690)。第二类,金刚烷胺(Amantadine)和金刚乙胺(Rimantadine)主要破坏流感病毒M2蛋白离子通道活性,能够抑制流感病毒的脱衣壳过程(Proceedings of the NationalAcademy of Sciences of the United States of America.2008,105,10967-10972)。然而,美国疾病预防控制中心抽样调查发现,2008/2009年的H3N2毒株和2009年大流行H1N1病毒中,100%的毒株对金刚烷类药物都具有耐药性;99.6%的季节性H1N1流感病毒对达菲具有耐药性
三萜类化合物是自然界中广泛存在的一类天然化合物,其结构包括A、B、C、D、E五个环,30个碳原子(Journal of the American Chemical Society,1996,35,8509-8509)。三萜类化合物由于其多种多样的生物及药理活性而引起越来越广泛的关注,如白桦脂酸及其衍生物已在临床试验中用作抗肿瘤和抗HIV的药物(U.S.Pat.Nos.5,679,828;6,689,767;6,369,109;U.S.App.Pub.No.2004/0204389);齐墩果酸是保护肝脏防止化学试剂损伤和防治HIV感染的有效成份(Journal of Natural Products.1998,61,1090-1095)。北京大学周德敏教授课题组首次发现了自然界广泛存在的五环三萜天然产物与不同的环糊精偶联具有很强的抗流感病毒进入的活性,并对其机制进行了深入的研究(Europe an Journalof Medicinal Chemistry.2017,134,133-139;Biomaterials.2016,78,74-85)。本发明三萜糖类衍生物以及其对流感病毒的抑制作用则未见报道。
发明内容
本发明提供了一类三萜-糖类衍生物,其化学结构式如下式所示:
其中,是单键或双键;
X和Y结合起来形成一个带有1-5个相同或不同取代基的五元环、六元环或七元环,所述取代基各自独立选自H,未取代的C1-C6烷基或被羟基、氨基或羧基取代的C1-C6烷基,未取代的C1-C6烷氧基或被羟基、氨基或羧基取代的C1-C6烷氧基,卤素,羧基,羟基,硝基,氰基,巯基,C1-C6硫烷基或NHR9’,所述R9’是H,未取代的C1-C6烷基或被羟基、氨基或羧基取代的C1-C6烷基;
R1是单糖、双糖、多糖、或者它们的衍生物;
所述单糖为葡萄糖、甘露糖、果糖、木糖、阿拉伯糖、半乳糖、核糖或脱氧核糖;双糖是麦芽糖、蔗糖或乳糖;所述单糖、双糖或多糖的衍生物是指单糖、双糖或多糖的1个、2个、3个或4个羟基被乙酰氧基、苄氧基或乙酰氨基取代。
R2和R7各自独立选自H,卤素,羟基,氰基,硝基,巯基,C1-C6硫烷基,未取代的C1-C6烷基或被羟基、氨基或羧基取代的C1-C6烷基,氨基,NR11’R12’,所述R11’和R12’各自独立选自未取代的C1-C6烷基或被羟基、氨基或羧基取代的C1-C6烷基;
优选地,R2选自H、OH、SH或NH2,更优选为OH。
优选地,R7选自H、OH、NH2或SH,更优选为OH。
R3、R4、R5、R6和R8各自独立选自H,未取代的C1-C6烷基或被羟基、氨基或羧基取代的C1-C6烷基;
优选地,R3、R4、R5、R6和R8各自独立选自甲基。
R9选自H,卤素,羟基,氰基,硝基,巯基,C1-C6硫烷基,羰基,肟基,未取代的C1-C6烷基或被羟基、氨基或羧基取代的C1-C6烷基。
本发明提供了一类三萜-寡糖偶联物,其中X和Y结合起来形成一个带有1-5个相同或不同取代基的六元环,所述取代基各自独立选自H,未取代的C1-C3烷基或被羟基、氨基或羧基取代的C1-C3烷基,羧基,羟基,硝基,氰基或NHR9’,所述R9’是H,未取代的C1-C6烷基或被羟基、氨基或羧基取代的C1-C6烷基。
所述X和Y结合起来形成一个带有1-5个相同或不同取代基的六元环的三萜-寡糖偶联物结构式如下:
其中R10,R11,R12,R13和R14各自独立选自H、OH、CH3、NHR9’,其中R9’是H,巯基,C1-C6硫烷基,未取代的C1-C3烷基或被羟基、氨基或羧基取代的C1-C3烷基。
进一步的R10,R11,R12,R13和R14各自独立选自H、OH、CH3、NH2。
进一步的R10,R11,R12,R13和R14各自独立选自H、OH、CH3。
进一步的R11和R12各自独立选自H或甲基。
进一步的R10是H。
本发明提供了一类三萜-寡糖偶联物,其中X和Y结合起来形成一个带有1-5个相同或不同取代基的五元环,所述取代基各自独立选自H,未取代的C1-C3烷基或被羟基、氨基或羧基取代的C1-C3烷基,羧基,羟基,硝基,氰基或NHR9’,所述R9’是H,未取代的C1-C6烷基或被羟基、氨基或羧基取代的C1-C6烷基。
所述X和Y结合起来形成一个带有1-5个相同或不同取代基的六元环的三萜-寡糖偶联物结构式如下:
其中R10,R11,R12和R13各自独立选自H、OH、-C(CH3)=CH2、NHR9’,其中R9’是H,巯基,C1-C6硫烷基,未取代的C1-C3烷基或被羟基、氨基或羧基取代的C1-C3烷基。
进一步的R10,R11,R12和R13各自独立选自H、OH、-C(CH3)=CH2、NH2
进一步的R10是-C(CH3)=CH2,R11,R12和R13各自独立选自H。
本发明另一目的是提供三萜-寡糖偶联物的具体结构,见下表;
本发明另一目的是将上述化合物应用在制备治疗或/或预防流感药物中的应用。
本发明的化合物能够用于预防或治疗流感,尤其是甲型流感;本发明化合物可阻断流感病毒进入细胞,但不仅局限于此机制。
本发明化合物可以以纯净化合物或化合物的混合物的形式给药,或者优选在药物赋形剂,稀释剂或载体中给药。
可以通过任何适当的途径来施用活性剂进行治疗。适当的施用途径可以包括口服,直肠、鼻、气雾或颗粒吸入剂,局部(包括含化和舌下),经皮、阴道、膀胱内、伤口内和胃肠外(包括皮下、肌内、静脉内、胸骨内、膜内、硬膜外和真皮内)。
本发明也涉及组合物,包含本发明化合物,与一种或多种药学可接受的添加剂和任选的其他药物一起。药学可接受的添加剂可以是载体,稀释剂,佐剂和(或)赋形剂的形式,可以包括所有常规的溶剂、分散剂、填充剂、固体载体、包衣剂、抗真菌或抗菌剂、皮渗透剂、表面活性剂等张剂和吸收剂,和缓释或控释基质。活性剂可以以适合同时,分开或连续施用活性剂的组分的试剂盒的形式;在与组合物的其他成分相容和患者生理耐受的意义上,每种载体,稀释剂,佐剂和/或赋形剂必须是“药学可接受的”。该组合物可以方便地以单元剂型的形式存在,可以通过制药领域公知的方法来制备;这些方法包括将活性成分与载体相混合的步骤,其中载体是由一种或多种助剂组成的;一般地,制备该组合物,包括将活性成分与液体载体、稀释剂、佐剂和/或赋形剂或精细分离的固体载体或两者均匀和直接地混合,然后如果必要使产物成型。
适合口服的本发明的组合物可以是以每个都包含预定量的活性成分的分离单元例如胶囊,囊剂或片剂的形式存在;作为粉末或颗粒;作为水相或非水液体中的溶液或混悬液;或者作为水包油性液体乳剂或油包水性乳剂;活性成分也可以以大丸剂,药糖剂或糊剂的形式存在。
可以通过任选与一种或多种助剂压片或成模来制备片剂;可以通过在适当的机器中压制自由流动形式例如粉末或颗粒的活性成分来制备压制片,任选与粘合剂(例如惰性稀释剂、防腐剂、崩解剂、淀粉羟乙酸钠、交联聚维酮、交联羧甲基纤维素钠),表面活性剂或分散剂混合。可以通过在适当的机器中将用惰性液体稀释剂湿润的粉末状化合物的混合物成型来制备模印片;任选可以将片剂包衣或刻痕,可以通过配制来缓释或控释活性成分,例如使用不同比例的羟丙基甲基纤维素来产生所需的释放性质;片剂任选可以具有肠溶衣,以在肠部分而不是胃中释放。
适合胃肠外施用的组合物包括水性和非水性等张无菌注射溶液,其可以包含抗氧化剂,缓冲剂,抑菌剂和使组合物与所预期的患者的血液等张的溶质;和水性和非水性无菌混悬液,其可以包括助悬剂和增稠剂。该组合物可以存在于单位剂量或多剂量的密封容器例如安瓿和管中,可以贮存在冷冻-干燥(冻干)条件下,仅需要在使用前加入无菌液体载体例如注射用水。可以由上述种类的无菌粉末,颗粒和片剂来制备无准备的注射溶液和混悬液。
适合局部施用于皮肤,即经皮施用的组合物可以包含溶解或悬浮在任何适当的载体或基质中的活性剂,可以是洗剂、凝胶、乳膏、糊剂、软膏等等的形式。适当的载体可以包括液状石蜡、丙二醇、蜡、聚氧乙烯和长链醇。也可以使用经皮装置例如贴剂,可以包含适当材料例如硝酸/乙酸纤维素,丙烯和聚碳酸酯制成的微孔膜。贴剂也可以包含适当的皮肤粘附性和基底材料。
本发明的活性化合物也可以以植入物的形式存在,其可以包含药物的聚合性装置,其中聚合物是生物相容性的和无毒性的。适当的聚合物可以包括水凝胶、硅酮、聚乙烯和生物可降解的聚合物。
本发明的化合物可以以持续(即控释)或缓释的形式施用。持续释放制剂是其中施用后活性成分在患者体内缓慢释放并在最小的时间里维持所需的药物浓度的制剂;持续释放制剂的制备是本领域技术人员公知的。剂型可以包括口服形式,植入物和经皮形式。对于缓释施用,活性成分可以作为例如,缓释颗粒悬浮或在脂质体内。
依据选择的化合物的特定活性,患者状况以及要处理的病症选择本发明化合物适合的剂量范围。本领域技术人员可以根据其普通知识和在本领域的经验适合的剂量范围。例如对于流感,人类适合的剂量范围可以为每人每天1-500mg,例如10-300mg,通常为30-150mg。
本发明的优点和技术效果如下:
1.本发明将三萜与寡糖偶联,弥补了三萜类水溶性差导致的成药性差的缺点,大大增强了三萜类化合物的抗流感病毒活性;
2.本发明中的三萜-寡糖偶联物的合成方法采用了CuAAC反应,生成三氮唑连接臂,相较于其他连接方式,不仅使反应变得快捷高效,还增强了化合物的稳定性;
3.本发明中的化合物以流感病毒进入细胞阶段为靶点,从源头上抑制了流感病毒感染,为抗流感病毒抑制剂的研究提供了依据。
附图说明
图1为噬斑抑制实验结果示意图;
图2为流感病毒的NP蛋白表达量检测结果示意图。
具体实施方式
下面通过实施例对本发明作进一步详细说明,但本发明的保护范围不局限于所述内容,实施例中方法如无特殊说明均采用常规方法,使用试剂如无特殊说明,均为常规市售试剂或采用常规方法配置的试剂。
定义
术语“C1-C3烷基”是指含有一到三个碳原子的烷基,例如甲基,乙基,丙基等。
术语“C1-C6烷基”是指含有一到六个碳原子的直链或支链烷基,例如甲基,乙基,丙基,异丙基,正丁基,异丁基,叔丁基,戊基或己基等。
术语“单糖”是指葡萄糖、甘露糖、果糖、木糖、阿拉伯糖、半乳糖、核糖或脱氧核糖。
术语“寡糖”是指麦芽糖、蔗糖或乳糖。
术语“衍生物”是指“单糖,寡糖,多糖”的1个、2个、3个或4个羟基被乙酰氧基,苄氧基或乙酰氨基取代;优选“单糖,寡糖,多糖”的一个羟基或2个,3个或4个羟基被乙酰氧基,苄氧基,甲氧基和/或苯甲酰氧基取代;或者“单糖,寡糖,多糖”的一个羟基被氢、氨基、乙酰氨基取代。
术语“三萜”是指由数个异戊二烯去掉羟基后首尾相连构成的物质,大部分为30个碳原子,少部分含27个碳原子的萜类化合物,例如齐墩果酸,白桦脂酸等。
术语“卤素”是指氟、氯、溴或碘。
术语“C1-C6硫烷基”是指其中一个氢原子被硫原子取代的C1-C6烷基。
实施例1:本三萜-寡糖偶联物的制备方法,以制备M8化合物为例,合成路线如下:
M8化合物的具体制备工艺如下:
(a)取1g L-鼠李糖于100mL反应瓶中,加入吡啶30mL溶解,此时将反应瓶置于冰浴中,用常压滴液漏斗缓慢滴加乙酸酐10.5mL,滴毕后移至室温反应,3h后薄层检测,展开剂石油醚:乙酸乙酯=1:1,CMC显色剂显色。直接蒸干吡啶与过量的乙酸酐,得化合物M30;粗产物可直接用于后续反应,无需纯化,但要保证薄层上产物点单一,若产物点不单一,则需经柱层析纯化;
(b)取1g化合物M30于100mL反应瓶中,加入30mL二氯甲烷溶解,置于冰浴中,用常压滴液漏斗滴加1.3mL的HBr乙酸溶液(33%),滴毕后移至室温反应,2h后薄层检测,展开剂比例石油醚:乙酸乙酯=1:1;采用水/二氯甲烷体系(水与二氯甲烷均为100mL)萃取,同时在分液漏斗中加入过量的无水碳酸钠,待没有气泡产生,取有机相蒸干,得无色油状液体M31 891mg,待用,产率93.8%;
(c)室温下隔夜搅拌反应,8h后薄层检测,展开剂比例石油醚:乙酸乙酯=1:1;蒸干DMF,采用水/二氯甲烷体系(水与二氯甲烷均为100mL)萃取1次,MgSO4干燥后柱分离,得白的固体M32 812mg,待用,产率88.5%;
(d)取1g齐墩果酸(OA)溶解于20mL DMF,分别加入TBTU 844mg和DIEA339mg,室温反应,12h后薄层检测,展开剂比例石油醚:乙酸乙酯=3:1;蒸干DMF,采用水/乙酸乙酯体系(水与乙酸乙酯均为100mL)萃取3次,取有机相于MgSO4干燥后蒸除溶剂,重结晶(乙醇:水=3:1);得絮状固体M25 1.6g,待用;
(e)取1g化合物M25用DMF溶解,加入121mg的炔丙胺,再加入279mg的碳酸钠,室温反应1h;TLC监测,展开剂石油醚:乙酸乙酯=3:1;柱分离纯化,洗脱条件石油醚:乙酸乙酯=3:1,得到白色固体化合物M26 1.2g,收率83%;
(f)取化合物M26 1g和化合物M32 460mg加入溶剂(二氯甲烷/水=1:1)后,再分别加入维生素C 401mg和五水CuSO4 507mg,室温快速搅拌反应8h后停止反应;TLC监测,展开剂石油醚:乙酸乙酯=1:1;直接将反应液倾入分液漏斗补加二氯甲烷和水进行萃取,取有机相蒸干,得白色粉末:1321mg,产率86.4%;
M8:1H NMR(600MHz,CDCl3)δ:0.52,0.77,0.86,0.87,0.89,0.98,1.14(7×CH3),2.00,2.10,2.13(3×CH3CO),0.52—2.36(m,other aliphatic ring protons),2.49(dd,J=3.54Hz,13.02Hz,1H),3.21(dd,J=4.2Hz,11.28Hz,1H),3.78-3.82(m,1H),4.34(d,J=5.58Hz,1H),4.36(d,J=5.58Hz,1H),4.47(d,J=5.16Hz,1H),4.50(d,J=5.22Hz,1H),5.16-5.22(m,2H),5.30(s,1H),5.38(t,J=3.24Hz,1H),5.65(dd,J=1.02Hz,2.7Hz,1H),6.09(s,1H),6.59(t,J=5.28Hz,1H),7.78(s,1H)13C NMR(150MHz,CDCl3)δ:15.45,15.69,16.60,17.64,18.37,20.67,20.77,20.89,23.55,23.71,23.94,25.87(2C),27.27,27.34,28.19,30.82,32.42,32.43,33.09,34.16,34.99,37.04,38.55,38.86,39.41,42.05,42.22,46.34,46.76,47.62,55.17,69.23,69.68,70.97,74.13,79.04,84.83,121.87,123.42,144.42,169.45,169.89,170.06,178.59;
(g)将化合物M8 1g置于50mL反应瓶中,用30mL甲醇溶解,再加入360mg甲醇钠,室温下搅拌1h,薄层色谱检测,二氯甲烷:甲醇=10:1,显色剂为茚三酮;蒸干溶剂后向反应瓶中滴加1N HCl 2mL,抽滤,取滤纸上的固体进行干燥,得白的粉末M15 857mg,产率93.6%;
M15:1H NMR(600MHz,CD3OD)δ:0.49,0.76,0.90,0.94,0.96,0.95,1.14(7×CH3),0.49—2.11(m,other aliphatic ring protons),1.37(d,J=5.94Hz,3H),2.77(dd,J=3.54Hz,13.14Hz,1H),3.13(dd,J=4.26Hz,11.1Hz,1H),3.47-3.56(m,2H),3.67(dd,J=3.18Hz,9.24Hz,1H),4.06(d,J=3.06Hz,1H),4.37(d,J=11.34Hz,1H),4.44(d,J=15.06Hz,1H),5.33(s,1H),5.95(s,1H),8.09(s,1H)13C NMR(150MHz,CD3OD)δ:16.11,16.48,17.68,18.32,19.65,24.16(2C),24.20,24.64,26.56,27.98,28.61,28.87,31.76(2C),33.68,33.91,34.29,35.19,35.79,38.25,39.94,39.96,40.71,42.73,43.00,47.70,47.75,56.80,72.45,73.23,74.85,77.11,79.79,88.57,124.37,124.74,145.13,180.61。
其余三萜-寡糖偶联物化合物制备方法同上,不同在于步骤d中三萜种类的不同及步骤f中寡糖的不同;其余化合物结构式(表1)和部分化合物的核磁共振1H及13C化学位移值如下:
M4:1H NMR(600MHz,CD3OD)δ:0.60,0.77,0.91,0.93,0.94,0.96,1.15(7×CH3),0.60—2.11(m,other aliphatic ring protons),2.79(dd,J=3.72Hz,13.38Hz,1H),3.14(dd,J=4.5Hz,11.34Hz,1H),3.50(dd,J=3.3Hz,9.72Hz,1H),3.56-3.63(m,2H),3.70-3.75(m,3H),3.77-3.83(m,3H),3.90-3.94(m,3H),4.36-4.46(m,3H),5.35(t,J=3.36Hz,1H),5.64(d,J=9.18Hz,1H),8.07(s,1H)13C NMR(150MHz,CD3OD)δ:16.01,16.39,17.63,19.48,24.04(3C),24.54,26.46,27.84,28.47,28.74,31.60,33.54,33.74,34.13,35.03,35.66,38.11,39.80,39.83,40.60,42.51,42.85,47.51,47.60,56.68,61.50,62.51,70.28,72.52,73.64,74.79,76.75,77.15,79.55,79.67(2C),89.46,105.11,124.09,124.21,145.06,145.92,180.54;
M11:1H NMR(600MHz,CD3OD)δ:0.54,0.77,0.90,0.91,0.94,0.95,1.14(7×CH3),0.54—2.11(m,other aliphatic ring protons),2.79(dd,J=3.84Hz,13.26Hz,1H),3.13(dd,J=4.38Hz,11.22Hz,1H),3.70(dd,J=3.36Hz,9.42Hz,1H),3.85(dd,J=0.84Hz,12.6Hz,1H),3.93-3.94(m,1H),4.00(dd,J=1.92Hz,12.66Hz,1H),4.12(t,J=9.24Hz,1H),4.36(d,J=15.12Hz,1H),4.44(d,J=15.06Hz,1H),5.33(t,J=3.42Hz,1H),5.45(d,J=9.12Hz,1H),8.02(s,1H)13C NMR(150MHz,CD3OD)δ:16.07,16.48,17.71,19.61,24.14,24.20,24.66,26.62,28.00,28.62,28.88,31.77(2C),33.71,33.90,34.31,35.19,35.88,38.27,39.96,39.98,40.72,42.69,42.97,47.63,47.73,56.84,70.36,70.93,71.55,74.99,79.84,90.74,123.45,124.35,145.22,146.32,180.55;
M12:1H NMR(600MHz,CD3OD)δ:0.56,0.77,0.90,0.92,0.94,0.96,1.15(7×CH3),0.56—2.12(m,other aliphatic ring protons),2.78(dd,J=3.54Hz,13.2Hz,1H),3.13(dd,J=4.5Hz,11.34Hz,1H),3.51-3.53(m,1H),3.73-3.79(m,3H),3.91(d,J=2.16Hz,1H),3.93(d,J=2.22Hz,1H),4.10(s,1H),4.34-4.46(m,2H),5.34(t,J=3.06Hz,1H),5.9(s,1H),8.16-8.17(m,1H)13C NMR(150MHz,CD3OD)δ:16.12,16.48,17.62,19.62,24.18(2C),24.68,26.62,27.99,28.61,28.88,31.76(2C),33.70,33.90,34.29,35.18,35.77,38.24,38.27,39.95,39.98,40.73,42.70,42.99,47.63,47.73,56.83,62.77,67.88,72.42,75.08,79.82,81.69,88.66,124.38,124.93,145.17,180.61;
M13:1H NMR(600MHz,CD3OD)δ:0.63,0.77,0.90,0.93,0.94,0.96,1.16(7×CH3),0.63—2.10(m,other aliphatic ring protons),2.79(dd,J=3.72,13.38Hz,1H),3.14(dd,J=4.62,11.46Hz,1H),3.47-3.58(m,3H),3.71(dd,J=5.52,12.3Hz,1H),3.83-3.88(m,2H),4.34(d,J=15.12Hz,1H),4.44(d,J=15.12Hz,1H),5.35(t,J=3.3Hz,1H),5.57(d,J=9.18Hz,1H),7.99(s,1H)13C NMR(150MHz,CD3OD)δ:16.13,16.49,17.79,19.63,24.03,24.18(2C),24.21,24.69,26.62,27.99,28.63,28.89,31.76,33.69,33.90,34.26,35.19,35.98,38.26,39.98,40.76,42.68,43.01,47.66,47.77,56.84,62.55,71.04,74.13,78.59,79.83,81.27,89.70,123.91,124.37,145.24,146.27,180.66;
M14:1H NMR(600MHz,CD3OD)δ:0.60,0.77,0.91,0.93,0.94,0.96,1.15(7×CH3),0.60—2.11(m,other aliphatic ring protons),2.80(dd,J=3.48Hz,13.26Hz,1H),3.47-3.92(m,12H),4.40(d,J=15.12Hz,1H),4.47(d,J=15.12Hz,1H),5.25(d,J=3.78Hz,1H),5.35(s,1H),5.67(q,J=8.46Hz,1H),8.22(d,J=10.2Hz,1H)13C NMR(150MHz,CD3OD)δ:15.98,16.35,17.65,19.47,24.02,24.04,24.51,26.45,27.81,28.47,28.73,31.59,33.53,33.73,34.12,35.00,35.31,38.10,39.78,39.81,40.59,42.46,42.82,47.53,47.55,56.67,61.69,62.70,70.75,71.48,73.67,74.13,74.87,75.03,77.99,78.24,79.68,79.71,80.08,89.96,102.94,124.21,124.82,144.99,180.72;
M16:1H NMR(600MHz,CDCl3)δ:0.67,0.78,0.88,0.89,0.90,0.99,1.16(7×CH3),0.67—1.80(m,other aliphatic ring protons),1.87(s,3H),2.03(s,3H),2.07(s,3H),2.09(s,3H),2.55(d,J=4.44Hz,1H),3.21(dd,J=4.08,11.22Hz,1H),5.25(t,J=9.9Hz,1H),5.4-5.46(m,3H),5.83(d,J=8.82Hz,1H),6.60(brs,1H),7.78(s,1H)13C NMR(150MHz,CDCl3)δ:15.49,15.67,16.73,18.38,20.29,20.65(2C),20.81,20.87,23.59,23.71,24.00,25.90,27.23,27.35,28.18,30.81,32.39,32.41,33.09,34.18,35.06,37.03,38.56,38.85,39.45,42.05,42.08,46.31,46.74,47.64,55.17,61.59,62.09,67.68,70.43,72.77,75.22,79.03,85.85,121.23,123.35,144.51,168.82,169.42,170.11,170.63,178.57;
M17:1H NMR(600MHz,CDCl3)δ:0.68,0.78,0.88,0.89,0.90,0.99,1.16(7×CH3),0.68—1.80(m,other aliphatic ring protons),1.88(s,3H),2.01(s,3H),2.05(s,3H),2.24(s,3H),2.56(dd,J=3.54Hz J=13.62Hz,1H),3.21(dd,J=3.84,11.16Hz,1H),4.11-4.15(m,1H),4.19-4.25(m,3H),4.64(dd,J=6.12Hz J=15.06Hz,1H),5.24(dd,J=3.36HzJ=10.26Hz,1H),5.41(t,J=3.36Hz,1H),5.54-5.57(m,2H),5.80(d,J=9.24Hz,1H),6.60(t,J=5.34Hz,1H),7.84(s,1H)13C NMR(150MHz,CDCl3)δ:15.47,15.67,16.66,18.38,20.36,20.62,20.75,20.80,23.58,23.69,24.04,25.90,27.24,27.34,28.17,30.80,32.37,32.39,33.09,34.18,34.99,37.03,38.55,38.84,39.43,42.03(2C),46.26,46.74,47.64,55.16,61.22,66.86,67.95,70.93,74.06,78.99,86.38,121.32,123.29,144.56,145.36,168.91,169.98,170.16,170.39,178.48;
M18:1H NMR(600MHz,CDCl3)δ:0.45,0.78,0.87,0.88,0.89,0.99,1.14(7×CH3),1.99,2.09,2.11,2.17(7×CH3CO),0.45—2.40(m,other aliphatic ring protons),2.55(d,J=9.48Hz,1H),7.26(d,J=9.48Hz,1H),3.94-3.97(m,1H),4.20(d,J=12.54Hz,1H),4.29(dd,J=5.4Hz,12.54Hz,1H),4.36-4.46(m,2H),5.28(dd,J=2.94Hz,10.08Hz,1H),5.35-5.40(m,2H),5.56(s,1H),6.17(s,1H),6.68(t,J=4.86Hz,1H),7.85(s,1H)13C NMR(150MHz,CDCl3)δ:15.42,15.62,16.45,18.25,20.56,20.70,20.73,20.80,23.60,23.96,25.75,27.06,27.19,27.27,28.14,30.74,32.23,32.44,33.03,34.13,34.95,36.95,38.54,38.80,39.30,42.00,42.02,46.27,46.69,47.54,55.16,62.14,64.90,68.83,70.97,75.63,78.98,84.67,122.13,123.35,144.34,144.83,169.31,169.62,169.91,170.64,178.69;
M19:1H NMR(600MHz,CDCl3)δ:0.63,0.77,0.87,0.88,0.89,0.98,1.15(7×CH3),0.68—1.80(m,other aliphatic ring protons),1.89(s,3H),2.04(s,3H),2.23(s,3H),2.58(dd,J=3.54Hz,13.08Hz,1H),3.21(dd,J=4.2,11.22Hz,1H),3.94(d,J=13.32Hz,1H),4.16-4.24(m,1H),4.66(dd,J=6.18Hz,15.06Hz,1H),5.24(dd,J=3.42Hz,10.08Hz,1H),5.39(t,J=3.3Hz,1H),5.43(s,1H),5.58(t,J=9.9Hz,1H),5.71(d,J=9.12Hz,1H),6.60(t,J=5.34Hz,1H),7.85(s,1H)13C NMR(150MHz,CDCl3)δ:15.41,15.68,16.66,18.37,20.37,20.70,21.08,23.54,23.69,23.94,25.90,27.22,27.33,28.17,30.80,32.38,32.52,33.10,34.17,34.92,37.03,38.53,38.84,39.40,41.94,41.98,46.23,46.67,47.63,55.16,67.29,67.76,68.26,70.63,79.01,86.82,121.34,123.21,144.48,145.25,169.00,170.05,170.35,178.40;
M20:1H NMR(600MHz,CDCl3)δ:0.66,0.78,0.87,0.89,0.99,1.16(7×CH3),1.87,1.98,2.06,2.07,2.09,2.11,2.17(7×CH3CO),0.66—2.30(m,other aliphatic ringprotons),2.54(d,J=12.72Hz,1H),3.18(s,1H),3.89-3.97(m,4H),4.09-4.17(m,3H),4.24(dd,J=4.8Hz,15.12Hz,1H),4.78(d,J=10.86Hz,1H),4.53(d,J=7.92Hz,1H),4.60(dd,J=6Hz,15.12Hz,1H),4.98(dd,J=3.42Hz,15.12Hz,1H),5.13(dd,J=7.98Hz,10.32Hz,1H),5.37(d,J=3Hz,1H),5.40-5.41(m,4H),5.77-5.79(m,1H),6.63(t,J=5.34Hz,1H),7.74(s,1H)13C NMR(150MHz,CDCl3)δ:15.58,16.58,18.26,18.85,20.23,20.51,20.63,20.65,20.68,20.72,20.80,22.82,23.48,23.59,23.93,24.98,25.78,27.01(2C),28.07,30.69,32.25,32.68,32.97,36.92,38.74,39.32,41.93,41.98(3C),46.17,46.63,47.35,47.52,55.05,60.83,61.72,66.58,69.01,70.54,70.82,70.91,72.61,75.55,75.85,78.88,85.52,101.11,121.26,123.27,144.41,145.20,168.94,169.11,169.58,170.07,170.13,170.20,170.41,178.54;
M21:1H NMR(600MHz,CDCl3)δ:0.66,0.78,0.87,0.89,0.98,1.16(7×CH3),0.66—1.80(m,other aliphatic ring protons),1.85(s,3H),2.02(s,3H),2.04(s,6H),2.07(s,3H),2.11(s,3H),2.14(s,3H),2.53(d,J=9.72Hz,1H),3.21(dd,J=3.96Hz,11.16Hz,1H),3.49(s,1H),3.98(dd,J=2.28Hz,9.48Hz,1H),4.06(dd,J=1.56Hz,12.36Hz,1H),4.14(t,J=6.54Hz,1H),4.23-4.27(m,3H),4.48(dd,J=1.92Hz,12.42Hz,1H),4.60(dd,J=6Hz,15.18Hz,1H),4.89(dd,J=3.9Hz,10.5Hz,1H),5.08(t,J=9.9Hz,1H),5.31-5.40(m,4H),5.44-5.47(m,2H),5.84(d,J=10.98Hz,1H),6.60(t,J=5.28Hz,1H),7.72(s,1H)13C NMR(150MHz,CDCl3)δ:15.51,15.68,16.73,18.38,20.32,20.71(5C),20.83,20.90,20.96,23.60,23.70(2C),24.04,25.89,27.25,27.35,28.18,30.81,32.38,33.09,34.19,35.05,37.04,38.57,38.86,39.45,42.06,42.11,46.31,46.75,47.64,55.17,61.55,62.62,68.00,68.85,69.32,70.11,71.07,72.48,75.25,75.42,79.03,85.38,96.00,121.34,123.38,144.54,145.38,169.14,169.56,170.07,170.13,170.44,170.66,170.69,178.65。
实施例2:本发明化合物抑制流感病毒进入细胞的生物活性评价方法
1、细胞病变(CPE)抑制试验
流感病毒感染细胞后会导致细胞病变,使得细胞活力降低;如果药物能够抑制流感病毒复制,则会降低细胞病变数量,提高细胞活力;具体方法如下:
(1)将犬肾上皮细胞(MDCK)以1:3的比例传代到白色的96孔板中,在37℃细胞培养箱中用含10%FBS的DMEM培养基培养24h;
(2)将流感病毒[A/WSN/33(H1N1),感染复数(MOI)=1]与100μM/L的待检化合物加入到100μl含有2μg/mL TPCK处理的胰酶,1%FBS的DMEM中,充分混匀;化合物的阴性对照为1%DMSO(稀释化合物所用的溶剂);同时设立一组只加各化合物不加病毒实验组,用来检测化合物对细胞活力的影响;
(3)将96孔板中的MDCK细胞的培养基吸出,将混合有病毒和化合物的培养基加入到MDCK细胞中,37℃细胞培养箱中培养48h。每个样品三个复孔;
(4)用CellTiter-Glo荧光细胞活性检测试剂盒(Cat.G7571,Promega)检测细胞活力,首先将细胞和CellTiter-Glo检测试剂放于室温环境,待其温度平衡至室温,将100μl/孔的CellTiter-Glo检测试剂加入到细胞的培养上清中,震动2min后,避光静置10min,用仪器TecanInfinite M2000PROTM检测细胞活力;
(5)EC50的计算方法:首先对化合物进行浓度系列稀释,然后利用上述方法测定出细胞活力;化合物对细胞病变的保护率=100×(1-(Test compound–Medi an Virus1)/(Median Cells-Median Virus2)),其中Test compound表示只加待检化合物不加病毒组的细胞活力;Median Virus1表示加了待检化合物和病毒组的细胞活力;Median Cells表示只加入1%DMSO组的细胞活力;Median Virus2表示加入1%DMSO和病毒组的细胞活力;将化合物浓度和相应的保护率输入到软件Prism,即可计算EC50;此方法已被广泛应用于抗病毒药物筛选领域;
(6)CC50的计算方法:CellTiter-Glo也可以用来检测化合物对细胞的毒性。首先对化合物进行浓度系列稀释,然后将其加入到细胞中,方法同步骤(2)-(4),但不加入病毒;培养48h后,测定细胞活力。然后将对照组细胞活力(1%DMSO)定义为100%,将其他各化合物组细胞活力标准化,除以对照组1%DMSO的细胞活力,再乘以100%。将化合物的浓度和相应的标准化的细胞活力输入到软件Prism,即可计算出CC50。
实验结果显示:与齐墩果酸相比,本发明化合物均表现出抗流感病毒活性,其中合物M7有非常好的抑制流感病毒活性,可以显著削弱病毒的感染性;被检测的化合物中OA、M2和M5有很强的细胞毒性,M1、M3、M4、M6和M9虽然在抗流感病毒活性没有M7明显,但与OA相比,细胞毒性有了显著的降低;其它化合物毒性都非常弱(见表1,2)。
表1在50μM浓度下,各化合物对MDCK细胞的毒性检测
化合物 | OA | M1 | M3 | M4 | M6 | M7 | M8 |
细胞活力(%) | 74.4 | 93.5 | 107.3 | 98.4 | 87.4 | 105.1 | 101.5 |
化合物 | M9 | M10 | M11 | M12 | M13 | M14 | M15 |
细胞活力(%) | 87 | 75.7 | 98.6 | 100 | 108.4 | 95.6 | 81.4 |
化合物 | M16 | M17 | M18 | M19 | M20 | M21 | DMSO |
细胞活力(%) | 101.9 | 79.8 | 94.9 | 109.3 | 81.2 | 85.3 | 100 |
表2在50μM浓度下,各化合物抗流感病毒活性,检测方法同表1
化合物 | OA | M1 | M3 | M4 | M6 | M7 | M8 |
病毒感染力(%) | 78.3 | 53.6 | 37.1 | 49 | 39.1 | 21.8 | 108.6 |
化合物 | M9 | M10 | M11 | M12 | M13 | M14 | M15 |
病毒感染力(%) | 36.9 | 66.7 | 66.8 | 67.9 | 74.4 | 76.2 | 67.3 |
化合物 | M16 | M17 | M18 | M19 | M20 | M21 | DMSO |
病毒感染力(%) | 76 | 67.8 | 66 | 74.8 | 76.1 | 69.7 | 104.7 |
通过CPE抑制试验证明化合物M7对流感病毒有着明显的抑制作用,强于阳性药物利巴韦林;CPE抑制试验表明M7的对流感病毒的EC50为36.3μM,而阳性药物达菲(磷酸奥司他韦,OSV-P)的EC50为40.8μM,利巴韦林(RBV)的EC50为50.1μM(见表3)。
表3 M7抑制流感病毒(WSN)的活性及其细胞毒性分析
2、噬斑抑制实验
利用噬斑抑制实验进一步印证化合物的抗病毒效果,具体方法如下:
(1)将MDCK细胞传代到12孔板中,在37℃细胞培养箱中用含10%FBS的DMEM培养基培养24h;使细胞密度达到0.4×106细胞/孔。用PBS清洗细胞一次;
(2)将A/WSN/33(H1N1)病毒(100PFU/孔)与系列稀释的化合物混合,稀释液为2μg/mL TPCK处理胰酶的DMEM。将混合液加入MDCK细胞中,置于37℃细胞培养箱中吸附1h;
(3)将病毒液吸出,用PBS清洗细胞三次,除去未吸附的病毒;
(4)用1mL含有1.5%低熔点琼脂糖,待检化合物,2μg/mL TPCK处理胰酶的无酚红DMEM覆盖细胞。注意温度不能过高,以免将细胞烫死;
(5)待4℃琼脂糖凝固后(10-15min)倒置放于在37℃培养箱培养;3-4天后对噬斑进行计数,计算病毒滴度。如果化合物对病毒有抑制作用,则噬斑数量会减少。
噬斑抑制实验表明M7对流感病毒的IC50<5μM(见图1与表4)。而M7在A549,MDCK以及293T细胞中的CC50均大于100μM,说明M7的细胞毒性很小。
表4噬斑抑制实验证明M7对于流感病毒有明显的抑制作用
M7浓度 | 100μM | 50μM | 25μM | 10μM | 5μM | 0μM |
噬斑数量 | 0 | 6.8±2.2 | 13.6±1.9 | 19.5±4.2 | 45.7±3.5 | 100 |
结果显示:流感病毒在MDCK细胞上可形成病毒噬斑,M7在5μM浓度下可以抑制一半以上的噬斑数量,即IC50<5μM。
3、加药时间点实验
用以分析化合物作用于病毒感染细胞的哪一阶段;具体步骤如下:
(1)将MDCK细胞传代到六孔板中,在37℃细胞培养箱中用含10%FBS的DMEM培养基培养24h;
(2)将A/WSN/33(H1N1)病毒(MOI=1)稀释到不含血清的DMEM中,感染MDCK细胞;
(3)流感病毒从吸附到子代病毒粒子释放,其复制周期约为6-8h;故在以下时间段将药物加入到细胞培养基中:0–10h,0–2h,2–5h,5–8h或8–10h;
(4)感染10h后,用冰预冷的PBS清洗细胞一次,用200μl/孔的PIPA裂解液裂解细胞。用细胞刮将细胞刮下,吸入1.5mL EP管中,置于冰上15min,以12,000rpm 4℃离心10min,将上清液转移到另一个1.5mL EP管中;
(5)吸取30μl样品与等体积的2×蛋白上样缓冲液混合,100℃煮样10min;
(6)将煮好的样品各20μl加入到12%的蛋白质凝胶加样孔中,进行SDS-PAGE电泳;
(7)用免疫印迹法(Western blotting)检测流感病毒的NP蛋白的表达水平(以此来检测病毒在细胞内的复制情况);同时以细胞蛋白GAPDH作为细胞内参(也可用于验证药物对细胞的毒性)。
通过上述加药时间点实验和上述血凝素实验可以初步断定,M7作用于病毒进入细胞过程,且干扰了病毒与细胞受体之间的结合(见表5和图2)。
表5加药时间点实验表明M7作用于病毒复制的早期(0-2h)
加药时间点 | 0-10h | 0-2h | 2-5h | 5-8h | 8-10h | DMSO |
病毒NP水平 | 0.26 | 0.38 | 1.0 | 0.98 | 1.0 | 1 |
结果显示,在全程给药(0-10h)以及0-2h加药均能够有效地抑制流感病毒的复制;说明药物在病毒感染后0-2h内发挥抑制作用,而在感染2h之后加药则无抑制效果;实验表明M7对HA有抑制作用,说明M7作用于病毒与细胞结合阶段。
4、假病毒实验
流感病毒假病毒实验用来验证化合物是否作用于病毒进入细胞阶段,以及化合物是否作用于其它高致病流感毒株,具有高度的安全性和可操作性。流感假病毒是一种重组的病毒颗粒,它的核心是来源于反转录病毒的基因组(除去包装基因的HIV基因组),而外层是流感病毒的囊膜蛋白血凝素蛋白(HA)和神经氨酸酶(NA);这种重组病毒能够像流感病毒一样感染细胞,但只能复制一次,不能够进行子代病毒包装。
假病毒的制备及化合物抑制假病毒感染实验的具体方法:
(1)将流感病毒的HA和NA基因克隆到真核表达载体pcDNA4/TO中,进行测序鉴定;
(2)利用质粒中提试剂盒(Promega)对质粒进行提取,利用分光光度计测定质粒浓度和纯度,用于下一步转染;
(3)将293T细胞传代到10cm细胞培养皿中,37℃细胞培养箱中培养24h,在质粒转染前1-2h对细胞进行换液;
(4)利用转染试剂lipofectamine2000(Invitrogen)将pcDNA4/TO-HA,pcDNA4/TO-NA与pNL4-3.Luc.E-R-载体各6μg共转染到293T细胞中,转染后4-6h换液,转染具体步骤见lipofectamine2000(Invitrogen)产品说明书;将转染后的细胞在37℃细胞培养箱中培养72h;
(5)流感假病毒粒子会被分泌到培养上清中,用0.45μM的滤器过滤含有假病毒的细胞培养上清,以除去培养基中的细胞和细胞碎片;
(6)将假病毒储存在-80℃低温冰箱中备用。VSV假病毒制备方法同上,唯一的区别是用表达VSVG的质粒取代流感病毒的pcDNA4/TO-HA和pcDNA4/TO-NA;
(7)将MDCK细胞传代到黑色透明底的96孔板中,在37℃细胞培养箱中培养24h;(8)将待检化合物与稀释过的假病毒进行充分混合,稀释液为含有2μg/mL TPCK处理的胰酶,1%FBS的DMEM;
(9)吸去96孔板中的细胞培养基,然后将100μl/孔的混合液加入到细胞中,在37℃细胞培养箱中培养48h;每个化合物三个复孔,每种化合物都包含一组VSV假病毒实验组,用来检测化合物对流感假病毒作用的特异性;
(10)利用Bright-glo萤光素酶检测系统(Promega)检测感染细胞中的萤光素酶(luciferase)的活性。首先将细胞培养板和检测试剂置于室温环境,将其温度平衡到室温;然后将100μl/孔检测试剂加入到96孔板中,震荡10s,避光静置2min;然后利用分光光度计测定luciferase活性;
所制备的假病毒基因组中缺失病毒复制所需的必要基因,所以假病毒丧失了复制能力,安全性高。除了A/WSN/33(H1N1)毒株外,我们还将选用的流感病毒毒株有A/VietNam/1203/2004(H5N1),因为此毒株为高致病性流感病毒毒株。这些毒株的HA和NA基因均可从北京义翘神州生物技术有限公司购买到,无需操作活病毒,所以实验是安全的。假病毒粒子中含有Luciferase报告基因,一旦进入细胞便可表达Luciferase基因,裂解细胞后加入酶的底物,酶标仪读数。
由于H5N1为高致病性流感病毒,所以本发明制备了H5N1和H1N1的假病毒,用来衡量M7抗病毒活性的广谱性。此种假病毒具有高度的安全性,可以在P2实验室进行操作。在M7浓度为50μM时,表现出对流感病毒H1N1和H5N1明显的抗病毒活性,抑制率分别为58.7%和14.3%(见表6);抑制率越高,检测到的相对萤光素酶活性就越弱。
表6假病毒实验表明M7能够抑制H1N1和H5N1流感病毒假病毒
假病毒是由HIV的核心蛋白和流感病毒的囊膜蛋白HA/NA组成;流感病毒的两种亚型H1N1和H5N1假病毒都被M7抑制;而M7对水疱性口炎病毒(VSV)假病毒没有明显的抑制能力;M7浓度为50μM;DMSO作为阴性对照,抑制率设为零。
Claims (4)
1.结构式如下式所示的三萜-寡糖偶联物:
其中,是单键或双键;
X和Y结合起来形成一个带有1-5个相同或不同取代基的五元环、六元环或七元环,所述取代基各自独立选自H,未取代的C1-C6烷基或被羟基、氨基或羧基取代的C1-C6烷基,未取代的C1-C6烷氧基或被羟基、氨基或羧基取代的C1-C6烷氧基,卤素,羧基,羟基,硝基,氰基,巯基,C1-C6硫烷基或NHR9’,所述R9’是H,未取代的C1-C6烷基或被羟基、氨基或羧基取代的C1-C6烷基;
R1是单糖、双糖、多糖、或者它们的衍生物;
R2和R7各自独立选自H,卤素,羟基,氰基,硝基,巯基,C1-C6硫烷基,未取代的C1-C6烷基或被羟基、氨基或羧基取代的C1-C6烷基,氨基,NR11’R12’,所述R11’和R12’各自独立选自未取代的C1-C6烷基或被羟基、氨基或羧基取代的C1-C6烷基;
R3、R4、R5、R6和R8各自独立选自H,未取代的C1-C6烷基或被羟基、氨基或羧基取代的C1-C6烷基;
R9选自H,卤素,羟基,氰基,硝基,巯基,C1-C6硫烷基,羰基,肟基,未取代的C1-C6烷基或被羟基、氨基或羧基取代的C1-C6烷基。
2.根据权利要求1所述的三萜-寡糖偶联物,其特征在于:单糖为葡萄糖、甘露糖、果糖、木糖、阿拉伯糖、半乳糖、核糖或脱氧核糖;双糖是麦芽糖、蔗糖或乳糖;所述单糖、双糖或多糖的衍生物是指单糖、双糖或多糖的1个、2个、3个或4个羟基被乙酰氧基、苄氧基或乙酰氨基取代。
3.根据权利要求2所述的三萜-寡糖偶联物,其特征在于,三萜-寡糖偶联物具体结构见下表:
4.权利要求1-3中任一项所述的三萜-寡糖偶联物在制备治疗或/或预防流感药物中的应用。
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